Prognostic and predictive markers are needed to predict the clinical outcomes of patients with advanced colorectal cancer (CRC) who receive standard first-line treatments. We performed a prospective cohort study in advanced CRC patients to identify a miRNA signature that could predict the benefit of receiving first-line chemotherapy for these patients. Twenty-one paired tumours and adjacent normal tissues were collected from advanced CRC patients and analysed by miRNA microarrays. Between tumour and normal tissues, 33 miRNAs were differentially expressed and was confirmed by qRT-PCR from another group of 67 patients from a prospective cohort study. A two-miRNA-based signature was obtained using the LASSO Cox regression model based on the association between the expression of each miRNA and the PFS of individual patients. Internal and external validation cohorts, including 40 and 44 patients with advanced CRC, respectively, were performed to prove the prognostic and predictive value of this signature. A signature was built based on two miRNAs, miR-125b-2-3p and miR-933. CRC patients were classified into low- and high-risk groups for disease progression based on this tool. The patients with low risk scores generally had better PFS than those with high risk scores. In the training set, the median PFS in the low- and high-risk groups were 12.00 and 7.40 months, respectively. In the internal validation set, the median PFS in the low- and high-risk groups were 9.90 and 5.10 months, respectively. In the external validation set, the median PFS in the low- and high-risk groups were 9.90 and 6.40 months, respectively. Furthermore, we detected miR-125b-2-3p associated with CRC cell sensitivity to first-line chemotherapy. Our two-miRNA-based signature was a reliable prognostic and predictive tool for tumour progression in patients with advanced CRC, and might be able to predict the benefit of receiving standard first-line chemotherapy in CRC. An estimated 1.4 million new cases of CRC are clinically diagnosed globally, resulting in 693,900 patient deaths from CRC in 20123. Over 20% of CRC cases are diagnosed at stage IV, an advanced stage, and higher percentages have been reported in developing countries5. Chemotherapy is one of the most common treatments for advanced CRC patients. FOLFOX (5-fluorouracil (5-FU), leucovorin and oxaliplatin)/XELOX  and FOLFIRI  are the first-line chemotherapeutic strategies for advanced CRC6. However, these similar chemotherapeutic strategies result in varying responses and different clinical outcomes in advanced CRC9. Moreover, primary and acquired drug resistance remain significant challenges to achieving satisfactory treatment effects. Thus, identifying effective biomarkers is essential for improving the prediction of treatment responses and guiding treatment decisions in advanced CRC patients.Colorectal cancer (CRC) is the third most common cancer diagnosed in both men and women and is the fourth leading cause of cancer-related death worldwide11. MiRNAs are involved in varied biological and pathophysiological processes, such as the cell cycle, cell differentiation, proliferation, apoptosis, etc.14. Apart from their important roles in biological processes, miRNAs also participate in a wide range of diseases, including many types of cancers16. Accordingly, miRNAs are studied as candidates for diagnostic and prognostic biomarkers and predictors of drug responses18. Several studies have demonstrated the roles of miRNAs as chemotherapeutic predictors in CRC21. Compared to a single biomarker, integrating multiple biomarkers into a single model can significantly improve the predictive value23.MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that typically induce gene silencing or mRNA degradation by binding to the target sites in the 3′ untranslated regions (UTRs) of their targeted mRNAsIn the present prospective observational study, we aimed to exploit and validate a multiple-miRNA-based signature using the least absolute shrinkage and selection operator (LASSO) Cox regression model to predict the progression-free survival (PFS) and response to controversial chemotherapeutic strategies in advanced CRC patients. We assessed the prognostic and predictive value of this signature in the training set, and then we validated this signature in two validation cohorts. Thus, our study identifies an effective prognostic and predictive biomarker signature for advanced CRC patients receiving standard first-line treatments.A total of 151 patients with histopathologically and clinically diagnosed advanced CRC were enrolled in the present study according to the selection criteria. The patients from SYSUCC were included and randomly assigned to the training set (67 patients) and internal testing set (40 patients), and another 44 patients from FPHFS were recruited as the external validation set. The clinicopathological characteristics of the patients in each set are shown in Table 24. A total of 178 upregulated miRNAs and 231 down-regulated miRNAs were identified in the tumour samples. Then, with the same data set, Cox regression analyses with a Wald threshold of p < 0.1 were run individually to screen the progression-related markers. A total of 33 miRNAs were identified as differentially expressed between the tumour and adjacent normal samples and were correlated with the patients’ PFS . Hierarchical clustering showed that the 21 pairs of tumour and adjacent normal tissues were separated into two discrete groups  were used to perform the entire LASSO Cox procedure, including optimal tuning parameter selection  and variable selection steps. In the present analysis, we obtained 1000 resamples and used different optimal λ parameters selected via 1-SE (standard error) criteria for each resample. We then calculated the RMIP for each miRNA. The RMIP is a measure of how likely a given miRNA is selected by the LASSO procedure if the dataset is perturbed. We observed continuing sharp decreases of RMIP until the third miRNA miR-5571-5p as ordered by the RMIP of each miRNA. As a result, two miRNAs, miR-125b-2-3p and miR-933, were selected for building a prognostic classifier . To confirm the prognostic accuracy of the two-miRNA-based signature, we conducted analyses using the internal testing and external validation sets. Similar results were observed in these two sets. In the internal testing set, the median PFS was 9.90 months (95% CI 6.89–12.91 m) for the low-risk group and 5.10 months (95% CI 3.57–6.63 m) for the high-risk group (p < 0.001). In the external validation set, the median PFS was 9.90 (95% CI 7.33–12.47 m) for the low-risk group and 6.40 months (95% CI 4.87–7.93 m) for the high-risk group (p = 0.002). For all the patients, the median PFS in the low- and high-risk groups was 11.30 (95% CI 9.36–13.24 m) and 7.20 months (95% CI 6.69–7.81 m), respectively (p < 0.001) ; 12.00% (3/25) for the low-risk group and 40.00% (6/15) for the high-risk group in the internal testing set (p < 0.001); and 17.86% (5/28) vs. 43.75% (7/16), respectively, in the external validation set (p < 0.001). For all patients, the progressive rate was 14.63% (12/82) for the low-risk group and 31.88% (22/69) for the high-risk one, respectively  Fig. . The prop < 0.05)  in the three cohorts respectively. The two-miRNA-based signature showed significantly higher prognostic value than any other single clinicopathological risk factor (05) Fig. .Fig. 3Tip = 0.004), in the internal testing set , and in the external validation set . These data suggested that this two-miRNA-based signature might be valuable for predicting the benefit of standard first-line chemotherapy in advanced CRC.We used multivariate Cox proportional hazards regression analysis to test the predictive value of the signature for first-line chemotherapy sensitivity in advanced CRC cases Table . The intTo explore the effect of miR-125b-2-3p and miR-933 on the activity of anticancer drugs, the MTS assay was performed in the CRC cell lines HCT116 and DLD-1. For each miRNA, specific mimics and inhibitors were used for functional experiments. The half maximal inhibitory concentrations (IC50) of anticancer drugs were calculated. As shown in Fig. Traditional clinicopathologic characteristics, such as gender, age, tumour size, tumour grade, and lymph node status, are rarely associated with the clinical response to chemotherapy of advanced CRC patients. However, the prediction of clinical response in metastatic CRC is vital for guiding therapeutic decisions. In the present study, we developed and verified a novel two-miRNA-based classifier to predict the disease progression and the benefit of receiving standard first-line chemotherapy in advanced CRC. Our results showed that this classifier could effectively assign advanced CRC patients into high- or low-risk groups with significant differences in PFS and that patients categorized as high risk based on the signature had worse survival outcomes after chemotherapy. Further use of this classifier might help identify advanced CRC patients who are most likely to benefit from first-line chemotherapy. Thus, the present miRNA signature for advanced CRC patients is a valuable prognostic and predictive tool, indicating that patients who are classified as low risk based on their risk scores have much lower chance of disease progression and have better responses to chemotherapy.28. In contrast, miR-125b is upregulated in leukaemia and prostate cancer, where this molecule seems to act as an oncogene30. These contradictory findings also occurred in CRC. On the one hand, downregulation of miR-125b was associated with lymph node metastasis in CRC, as shown by significantly increased cell invasion, migration, and MMP activity31. On the other hand, high levels of miR-125b expression in CRC were associated with poor survival32. In the present study, miR-125b-2-3p was down-regulated in CRC tumour tissues, and the expression of this molecule was positively correlated with PFS, which was consistent with the previous metastatic CRC study31.There were plenty of miRNAs which were identified differentially expressed in CRC compared with normal tissues. Among these miRNAs, two miRNAs enrolled in the signature, miR-125b and miR-933, have previously been reported as playing potential roles in cancer. MiR-125b acts as either an oncogene or tumour suppressor and shows heterogenic expression in different carcinomas. For example, miR-125b is down-regulated in ovarian, bladder and breast cancer, which suggests a tumour suppressor function33. However, there was no study to compare the expression of miR-125b-2-3p in stromal cells and in cancer cells. Due to lack of microdissection procedure, our study cannot exclude the effect of stromal cells in CRC, so further study is needed to explore the role of miR-125b-2-3p in stromal cells.Moreover, our data showed that miR-125b-2-3p affected CRC cell sensitivity to chemotherapeutic therapy. The effects of miR-125b-2-3p knockdown were more notable than those of its overexpression in CRC cells since the expression of miR-125b-2-3p was high in these two CRC cell lines. On the other hand, as we known, stromal cells play important roles in CRC biology and affect the response to chemotherapy. It was reported that the expression of miR-125b-2-3p in CRC stroma has no significant difference with that in the epitheliaIn our study, the miR-933 knockdown or overexpression cells had no significant change in their response to the anticancer drugs. Furthermore, the Cox regression analysis showed that there was no significant association between the expression of miR-933 and CRC patients’ PFS, which might mean that the miR-933 is not an independent prognostic and predictive biomarker in CRC.36, although further mechanistic exploration is needed.The function and mechanism of microRNA-933 in CRC tumourigenesis, progression, and response to chemotherapy has not been thoroughly investigated. However, several studies exploring single-nucleotide polymorphisms in miR-933 have shown that this molecule is associated with susceptibility to several types of human cancersHowever, our study was a prospective observational study and not a clinical trial; therefore, there may be some inherent biases of such a study format. Moreover, the patients enrolled in the present study were all Chinese individuals whose clinical characteristics might be different from those of other ethnic groups; thus, the generalizability of this study might be limited. Clearly, an open-label, multicentre, randomized clinical trial is needed to further validate this novel signature. Furthermore, the mechanism for how miR-125b-2-3p affects CRC sensitivity to chemotherapy warrants further exploration.In summary, the present study shows that the two-miRNA signature can successfully classify advanced CRC patients into groups with high and low risks of tumour progression. In addition, this signature might be a reliable prognostic and predictive tool for tumour progression in patients with advanced CRC and might be able to predict the benefit of receiving standard first-line chemotherapy in CRC.This prospective observational study was started in 2008. A total of 151 patients with histopathologically and clinically diagnosed advanced CRC from 1 January 2008 to 30 June 2014 were enrolled in this project. The following were the criteria for enrolment: (1) patients diagnosed with advanced CRC; (2) histologically confirmed CRC with at least one measurable lesion as defined by the Response Evaluation Criteria in Solid Tumours; (3) patients who received National Comprehensive Cancer Network (NCCN)-guided first-line chemotherapy (FOLFOX or FOLFIRI strategy); (4) patients with completed clinicopathological information and follow-up data; (5) patients have not received previous chemotherapy for the target lesions, however, patients who had received adjuvant/neoadjuvant chemotherapy were enrolled if the interval was more than 12 months after the end of chemotherapy; and (6) sufficient formalin-fixed paraffin-embedded (FFPE) tissues for further study. Prior to primary chemotherapy, tumour samples were acquired during palliative operations or colonoscopy biopsies and embedded in paraffin.For the training and internal testing sets, 107 metastasis CRC patients treated at Sun Yat-sen University Cancer Center  were included from 1 January 2008 to 30 June 2014. These 107 patients were randomly divided into training and internal testing sets . To verify the prognostic or predictive accuracy of the signature, we included another 44 advanced CRC patients as the independent external validation cohort from the Foshan First People’s Hospital (FFPH), Foshan, between 1 December 2008 to 31 December 2013. The same inclusion and exclusion criteria were used. Follow-up procedure for these patients was finished on 31 July 2016. The median follow-up time was 44.6 month. Additionally, to obtain the miRNA expression profiles, 21 frozen tumour and paired adjacent normal mucosa samples from metastatic CRC patients obtained at SYSUCC from 1 January 2003 to 31 December 2005 were used.The clinical and clinicopathological classification and stage were clarified according to the American Joint Committee on Cancer (AJCC) criteria. Prior to carrying out the research, this project was approved by the Institutional Research Ethics Committee and all the participants signed informed consent forms. All the patients received tumour assessments every 6 weeks during therapy and at the end of treatment. PFS was defined as the period between the initiation treatment of first-line chemotherapy and the date of disease progression or death. The blinded endpoint study was not applicable in this project. The study was followed REMARK guideline .The FFPE tissue samples were composed of at least 80% tumour cells. Total RNA was isolated from 151 metastatic CRC samples and 21 paired CRC tumour and adjacent normal tissues as previously described38. Briefly, total RNA was purified and concentrated using the mirVana™ miRNA Isolation Kit . Purified RNA was labelled with fluorescein Cy3 and hybridization was conducted using an Agilent Human miRNA Microarray (miRBase release 19.0). The fluorescence intensities were converted into digital data and Log2 transformed using Feature Extraction (version 10.7). Differential gene expression was analysed by using GeneSpring software version 12.0 . The miRNAs were categorized as significantly differentially expressed if the p value was lower than 0.05. Heat maps were formed using the Cluster 3.0 package software. Microarray data were deposited into the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI) (accession no. GSE108153).RNA labelling, hybridization and array scanning were carried out as previously described and performed by CapitalBio MiRNA expression was detected by qRT-PCR in the 151 CRC samples and 21 paired CRC and adjacent normal tissues. Total RNA was obtained with TRIzol reagent , and 2 μg total RNA was reverse transcribed using the All-in-One™ miRNA First-Strand cDNA Synthesis Kit . The All-in-One™ miRNA qPCR Kit  and Roche Lightcycler 480 instrument  were used for qRT-PCR analysis. All experiments were conducted according to the manufacturer’s instructions. The results were normalized to U48 expression and analysed using in the 2-Δct method. The primers were synthesized by GeneCopoeia, Inc.p < 0.1).We performed the miRNA microarray on 21 paired CRC cancer and adjacent normal frozen tissues samples. We identified the differentially expressed miRNAs using the significance analysis of microarrays with a false discovery rate (FDR) < 0.05. By combining the RNA expression data from the microarray and follow-up data, we obtained 33 significantly differentially expressed miRNA candidates correlated with PFS by Cox regression analysis  analysis, which was measured by the area under the curve.To determine the miRNAs most predictive of a high/low recurrence risk signature, we utilized the LASSO bagging strategy. At the data preparation step, we first obtained 33 candidate markers from the differential expression and uni-variable Cox regression analysis described above, and we then examined their RNA abundance in a training data set of 67 cancer samples using RT-PCR. Since the relative RT-PCR values of miRNA in each sample were too small to manage, we applied logarithmic transformation (2 bases) on 10,000 multiplied matrices to normalize the values into a readable range. As a result, a matrix of 33 variables and 67 data points (samples) was built for further LASSO bagging analysis, the procedure for which is described as follows: (1) the data points were resampled 1000 times with replacement to generate 1000 training matrices. (2) For each matrix with recurrent survival outcomes, we performed LASSO Cox regression analysis using 10-fold cross validation. The tuning parameter λ was selected by 1-SE (standard error), and we finally obtained a list of variables that had beta-coefficients different than zero in LASSO. (3) All variable lists obtained in each resample were combined to calculate the resample model inclusion proportion (RMIP) for each miRNA . (4) The RMIP was used as the weight of each variable, and the top two miRNAs were selected as candidate markers for building the signature. All running analysis scripts were programmed with R (v3.3.1), and LASSO was adopted from the glmnet package (2.0-10).2. All cell lines were tested and authenticated by short tandem repeat DNA testing at Beijing Microread Gene Tech. Co., Ltd.  prior to use.The HCT116 and DLD1 CRC cell lines were purchased from ATCC  and cultured in RPMI1640 basic medium (1 × ) with 10% foetal bovine serum  at 37 °C with 5% CO3 cells/well and incubated with irinotecan, fluorouracil and oxaliplatin  for 72 h. Then, the cells were stained with 20 μl of sterile MTS  -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl)-2H-tetrazolium) dye  for 2 h at 37 °C. The absorbance was measured at a wavelength of 490 nm. All experiments were conducted in triplicate.Cells were seeded onto 96-well plates at a density of 3 × 10t test was used to compare continuous variables between paired tumour and adjacent normal tissues. For survival analyses, the curves were plotted with the Kaplan-Meier method, and the different groups were compared using the log-rank test. Adjusted hazard ratios (HRs) with 95% confidence intervals (CI) were calculated using Cox proportional hazards modelling. The median value of the risk score in the training set was taken as the cut-off value. Post hoc statistical power analysis was calculated to evaluate the study sample size. All statistical tests were two-sided and considered significant at two-sided p values < 0.05, unless specifically stated. All the data analyses above were performed using SPSS version 22.0 statistical software or R3.1.2  software.Student’s Supplementary Figure LegendsSupplemental Figure S1Supplemental Figure S2Supplemental Table 1Supplemental Table 2


Computational RNA 3D structure prediction and modeling are rising as complementary approaches to high-resolution experimental techniques for structure determination. They often apply to substitute or complement them. Recently, researchers’ interests have directed towards in silico methods to fit, remodel and refine RNA tertiary structure models. Their power lies in a problem-specific exploration of RNA conformational space and efficient optimization procedures. The aim is to improve the accuracy of models obtained either computationally or experimentally.http://rnafitme.cs.put.poznan.pl/Here, we present RNAfitme, a versatile webserver tool for remodeling of nucleobase- and nucleoside residue conformations in the fixed-backbone RNA 3D structures. Our approach makes use of dedicated libraries that define RNA conformational space. They have been built upon torsional angle characteristics of PDB-deposited RNA structures. RNAfitme can be applied to reconstruct full-atom model of RNA from its backbone; remodel user-selected nucleobase/nucleoside residues in a given RNA structure; predict RNA 3D structure based on the sequence and the template of a homologous molecule of the same size; refine RNA 3D model by reducing steric clashes indicated during structure quality assessment. RNAfitme is a publicly available tool with an intuitive interface. It is freely accessible at RNAfitme has been applied in various RNA 3D remodeling scenarios for several types of input data. Computational experiments proved its efficiency, accuracy, and usefulness in the processing of RNAs of any size. Fidelity of RNAfitme predictions has been thoroughly tested for RNA 3D structures determined experimentally and modeled in silico. RNAs play a central role in all aspects of cell functioning. They constitute genomes of viruses and retroviruses. They have also been recognized as new targets in RNA diagnostics, molecular therapy, and nanotechnology. Biological functions and physicochemical properties of RNAs depend on their 3D structure and dynamics. The details of RNA structures have been mostly uncovered using high-resolution experiments of X-ray crystallography, NMR or electron cryo-microscopy. However, recent decades have resulted in the development of computational methods that substitute or complement experimental techniques and facilitate processing of their output data. Among them are the tools for RNA 3D structure prediction and modeling which have been applied in such fields as molecular and structural biology, RNA therapeutics or engineering of biomaterials .Generally, predictive algorithms have located in one of two groups: (i) physics-based methods which proceed with de novo prediction, using dynamics simulation of a full-atom or coarse-grained RNA models; (ii) knowledge-based techniques that apply various structure templates in homology modeling. Until today, several methods have been released and acknowledged pretty reliable under these two groups –9. SinceRNA structure modeling usually relies on computer-aided manipulation of the 3D models determined experimentally or predicted computationally. To varying degrees, it requires the user intervention that consists of different operations like removal, insertion, replacement, rotation or shifting of the tertiary structure fragments. Total or partial automation of the modeling process can be possible thanks to the use of problem-tailored optimization procedures, well-defined conformational constraints and structure data repositories. The latter ones constitute the search space for combinatorial algorithms which look for optimal conformation of analyzed RNA.i, ε i, ζ i, α i + 1, βi + 1, γ i + 1, δ i + 1 angles, where i, i + 1 denote the i-th and the (i + 1)-th nucleotide in the RNA chain. This concept has been applied to construct the library of RNA backbone conformers which encompassed nearly 50 discrete suites essentially free of steric clashes [In contrast to proteins, the extent of possible RNA conformations is much complex. The RNA backbone is defined by six degrees of freedom. Thus, exploring RNA conformational space and constructing its components is a non-trivial task. An initial solution to this problem has proposed to build conformer libraries founded on torsion angles. Typically, α, β, γ, δ, ε, and ζ dihedral angles of sugar-phosphate backbone specify individual nucleotide unit of RNA . An alte clashes . Modelin clashes . However clashes . The con clashes , althougIn our previous work , we haveHere, we present RNAfitme, a versatile webserver tool for modeling of nucleobase and nucleoside residue conformations in fixed-backbone RNA 3D structures. It allows modeling, reconstructing, and remodeling from one to all residues in the RNA chain. RNAfitme method has adapted a graph-based algorithm to look for the optimal mutual arrangement of nucleobase/nucleoside residues in the RNA structure. The most important criterion followed by the algorithm is the minimization of steric clashes between predicted and fixed structure fragments. Several libraries  applied The webserver has appeared particularly well suited to the following applications. First is a reconstruction of full-atom RNA 3D model from the fixed-backbone atom coordinates. It is accompanied by the restoration of RNA secondary structure. In this application, RNAfitme allows studying what 3D structures can be reconstructed on a given sugar-phosphate backbone. Next, the tool can be applied to precisely remodel – in a given RNA structure – selected nucleobase/nucleoside residues pointed by the user. The latter application allows observing the influence of local modifications on the secondary and the tertiary structure of analyzed RNA. The third aspect concerns template-based prediction of RNA 3D model with a homologous RNA sequence(s) of the same size. This additional capacity of RNAfitme is restricted to cases which can be solved using substitutions only (insertions and deletions are not supported). Finally, let us add that in any usage scenario, the tool optimizes RNA 3D structure model by reducing steric clashes.http://rnafitme.cs.put.poznan.pl/.RNAfitme is free and open to all users without any login requirement. The webserver is publicly available at RNAfitme aims to model – in a fully-automated way – nucleobase and nucleoside residues in RNA 3D structure with preserving fixed coordinates of sugar-phosphate backbone atoms. The system kernel is a collection of conformer libraries and a graph-based optimization algorithm that builds the preliminary RNA 3D model. The algorithm’s tasks include: searching the library for candidate conformers; conformers’ reconstruction on the fixed-backbone; selecting the subset of promising candidates; controlling the reconstruction accuracy from a global perspective. The libraries create a discrete conformational space explored by the candidate search procedure. Additionally, RNAfitme includes a pre-processing module that validates and cleans an input data, and a post-processing one to execute energy minimization thus, improving the geometry of the preliminary 3D structure. In the latter step, the minimization encompasses only these structure fragments that were remodeled in RNAfitme. It is conducted using CHARMM force field by NAMD method . Next seAt the input, the user should upload PDB file that includes either full-atom RNA 3D structure or sugar-phosphate backbone atoms only, or give a PDB identifier of the structure to be remodeled. In the latter case, the PDB file is automatically downloaded from the Protein Data Bank . DependiFirst, the input 3D structure is cleared of atoms that are not compatible with the PDB format. Next, a procedure checks if the required atoms are present in the input data. In the nucleoside mode the input 3D structure should include backbone atoms . In the nucleobase mode – backbone and sugar ring atoms . All the remaining atoms of remodeled/reconstructed fragments are removed from the input structure. The pre-processing module also verifies if the sequence length (if given) and input RNA chain length are equal. The lower bound for the input data size has been set to 3 nucleotide residues. No upper bound is defined. Finally, from input coordinates, the system computes lengths of covalent bonds, values of torsional angles along the backbone, and pseudotorsion angles. This data is used by optimization algorithm in further processing.RNAfitme has a built-in repository which consists of five conformer libraries. These are: (i) Neural gas clustering - Euclidean distance - High-resolution RNA structures (NG-E-HR); (ii) Neural gas clustering - MCQ distance - High-resolution RNA structures (NG-M-HR); (iii) Neural gas clustering - Euclidean distance - 23S rRNA (NG-E-23S); (iv) K-medoids clustering - Euclidean distance - all RNA structures (KM-E-ALL); (v) Neural gas clustering - Euclidean distance - all RNA structures (NG-E-ALL). Their construction has been described in detail in . By defaHR set). In the case of NG-E-23S, the crystal structure of the Haloarcula marismortui ribosomal subunit (PDB ID: 3CC2) [23S set). For KM-E-ALL and NG-E-ALL, we have taken all RNA 3D structures (ALL set) deposited in the Protein Data Bank [HR set included 65,134 residues, the 23S set – 2876, and the ALL set – 1,743,940. Next, the residues in every set were hierarchically clustered using either neural gas (NG) [In general, each library was generated following the same protocol . In everD: 3CC2)  was chosata Bank  at that gas (NG)  or k-medgas (NG)  algorithgas (NG) . Finallygas (NG) . Five li0, τ1, τ2, τ3, τ4, χ), coordinates of all atoms, and lengths of covalent bonds.Every library has a different size and hierarchical structure. NG-E-HR contains 282 groups resulting from 1st-stage clustering . They include 1742 elements resulting from 2nd-stage clustering (based on the χ angle). For the remaining libraries, these numbers are as follows: 58/319 in NG-M-HR, 231/580 in NG-E-23S, 16/32 in KM-E-ALL, and 381/1637 in NG-E-ALL. Detailed information about residue types representation in each library is included in . Each coThe RNAfitme method has been inspired by the approach proposed for proteins . Our optFor every reconstructed/remodeled residue in the nucleoside mode, the most similar ribose ring conformers are looked for in the selected library. Their adjustment to the fixed-backbone is assessed based on MCQ score  computedTo optimize further computation, the number of candidates is reduced to a maximum of ten for every residue. In each case, the nucleobase/nucleoside residue is reconstructed on the fixed-backbone based on the torsional angle-driven characteristics of a conformer. Next, RMSD (Root Mean Square Deviation)  score iscm that remained in the conformational search space. First, the energy Efixed(cm) between cm and the fixed part of the input structure is computed. Second, for each yet unresolved conformer cn, the method estimates interatomic energy Epair. In all the cases, the energy is approximated using Lennard-Jones potential [Two types of energy terms are calculated for every promising candidate otential .cm(ri) found for residue ri can be pruned from the search space if there is another conformer cn(ri) such that it always contributes lower energy with all other neighboring conformers present in the search space than cm(ri). DDE is applicable in cases when more than one candidate conformer is considered for a single nucleobase/nucleoside residue.In this step, the dead-end elimination (DDE) procedure  is perfoG. Every residue ri from the considered set corresponds to a node vi in graph G. Two nodes vi, vj∈G are connected with an edge if there exists at least one pair of conformers cm(ri), cn(rj) for which a pairwise interaction energy is positive: Epair(cm(ri), cn(rj)) > 0.At this stage, only these residues are considered for which more than one conformer is present in the conformational search space. Such residues are taken to create a residue-residue interaction network which has a form of an undirected graph K will denote the number of identified connected components. A connected component in an undirected graph G is defined as a subgraph G’⊂G in which there is a path between any two nodes of G’ and which is not connected to any other node that does not belong to G’.In this step, a procedure runs to find all connected components in the residue-residue interaction network. Gk’ of G (k = 1..K). It decomposes Gk’ into a block-cut tree . Next, articulation points  are identified in every subgraph Gk’.A depth-first search algorithm is performed on every connected component At this stage, we find the minimum energy for each connected component. It is done while a branch-and-bound backtracking algorithm performs to solve every block-cut tree. As a result, each articulation point is associated with a subset of conformers selected for the corresponding residues and their total interatomic energy. Articulation points are then recursively collapsed, and their energy propagates further. The process stops when no articulation point exists.The final stage of processing in the RNAfitme system is to minimize the repulsive steric energy of the preliminary 3D model. This step may be omitted if the user unchecks the “Minimize 3D model energy” option. Although the final minimization is not mandatory, its application is suggested. Therefore, the option is enabled by default.The energy minimization procedure is conducted by NAMD, using CHARMM force field . NAMD imAt the output, the user obtains an RNA three-dimensional structure model in the PDB file format. The model is visualized using JSmol which has been incorporated into the RNAfitme system. In the visualization, fixed atoms are dark blue, while the remodeled ones are green. Visualization panel allows for interactive manipulation. The resulted 3D model can be downloaded and saved in various file formats . A popup menu to allow such operations shows up after clicking the right mouse button in the panel area. The log file is provided along with the output 3D model. It enumerates all computing steps performed by RNAfitme and shows the result of every intermediate step. Computing time is also given.All the presented output data can be downloaded in a zipped archive by selecting the checkbox and clicking “Download selected results”. The result page also allows to run RNAfitme once again for the same RNA 3D structure with input parameters changed. This is possible after clicking “Run with different parameters” button and selecting the other set of input parameters.In this section, we present and discuss the results of RNAfitme experimental runs in several usage scenarios and for selected examples of RNA structures. We analyze the results given by the webserver in the case of structure processing in nucleobase and nucleoside modeling mode, and we confirm the accuracy of obtained 3D models under global quality assessment.Aspergillus restrictus  as a reference [In the first computational experiment, we run RNAfitme to reconstruct full-atom 3D structure based on the fixed-backbone and RNA sequence provided by the user. For this experiment, we selected crystal structure of a 31-mer SRD RNA inhibitor bound to the ribotoxin restrictocin from eference . We prepeference  to checkeference , 41 run The second experiment aimed to remodel the 3D structure to correct it by reducing steric clashes. We selected glutamine tRNA bound to the cognate synthetase   as an exIn the third single-case experiment we run RNAfitme to remodel user-selected nucleoside residues in the input RNA 3D structure. We chose the crystal structure of cysteine tRNA bound to the cognate synthetase   as exampHaloarcula morismortui ribosomal subunit (PDB ID: 1S72) [The fourth experiment aimed to check how RNAfitme method performed for non-canonical base pairs, and mutations of base pairs introduced into the structure. As a test example, we selected the kink-turn motif 23S KT-7 cut out from D: 1S72) . This reD: 1S72) , is dispD: 1S72) , S/S traD: 1S72) .Fig. 4ThS19 of representative RNA 3D structures deposited in the Protein Data Bank [S19 was uploaded to RNAfitme input and processed in both, nucleobase and nucleoside remodeling mode, with NG-E-HR library and energy optimization option selected. Next, we compared the output 3D models with the original structures to find if the MolProbity-reported steric clashes were reduced. We also investigated the extent to which the tertiary and the secondary structure changed. In the first case, we analyzed RMSD of the output model with respect to the input (reference) structure. For the nucleobase remodeling mode, RMSD was computed over the following atoms: C2, C4, C5, C6, C8, N1, N2, N3, N4, N6, N7, N9, O2, O4, O6. For the nucleoside remodeling mode, the subset of considered atoms included: O4’, C1’, C2’, O2’, C3’, C4’, C2, C4, C5, C6, C8, N1, N2, N3, N4, N6, N7, N9, O2, O4, O6. To analyze the secondary structures, we used their extended dot-bracket representations that encoded canonical base pairs only. The secondary structures were annotated using RNApdbee 2.0 [S19 dataset, and we analyzed the accuracy and quality of the resulting RNA 3D models. For every library, we computed average values of RMSD, INF and Clash score : Platform independent.Programming language: Java 1.7.0, Spring.io.Any restrictions to use by non-academics: no restrictions.


Cynanchum wilfordii (CW) ethanolic extract on blood cholesterol levels in adults with high low-density lipoprotein cholesterol (LDL-C) levels. In a double-blind, randomized, placebo-controlled, parallel trial, 84 subjects were recruited. Participants were randomly divided into two groups with a low-dose (300 mg/d) or high-dose (600 mg/d) of CW. Levels of very low-density lipoprotein (p = 0.022) and triglycerides (p = 0.022) were significantly lower in the low-dose CW group than in the placebo group after 8 weeks. In a subgroup of participants with LDL-C≥ 150 mg/dL (n = 33), there was a significant decrease in total cholesterol , apolipoprotein B , and cholesteryl ester transfer protein  after 8 weeks of CW. The correlation between changes in total cholesterol and baseline LDL-C levels was significant in the groups that received both doses of CW . These results show that the CW ethanolic extract can regulate blood cholesterol in subjects with LDL-C≥ 150 mg/dL.We evaluated the effects of  Cholesterol is an essential component of the body. It influences the fluidity of the cell membrane and is a precursor to steroid hormones. One-third of the total cholesterol (TC) in circulation is derived from food through an exogenous cholesterol transport pathway, and two-thirds is synthesized in the body via an endogenous pathway . CholestDespite this tight regulation, more cholesterol than needed can accumulate in blood vessels causing many diseases. Hyperlipidemia has been associated with high levels of LDL-C and low levels of HDL-C. The number of subjects with hyperlipidemia has been increasing due to diets that are high in saturated or trans-fat. Hyperlipidemia can also result in biliary obstruction, chronic kidney disease, type 2 diabetes mellitus, high blood pressure, and hypothyroidism. Elevated levels of LDL-C can lead to a buildup of plaques within the arteries and increase the risk of cardiovascular disease (CVD), which is a major cause of death due to myocardial infarction and stroke . It was Cynanchum wilfordii (CW) is found throughout Northeast Asia and its roots have been used in traditional medicines [p-hydroxyacetophenone [edicines . In Koreedicines . Its immedicines . The aquedicines . CW has edicines . The comophenone . Cynandiophenone . Cynandiophenone ,18. Caudophenone . Caudatiophenone . Caudatiophenone .−/− mice fed a high-fat and high-cholesterol diet [Previous studies have reported that CW reduces fat accumulation and liver damage caused by high-fat and high-fructose diets in rats by suppressing COX-2, NF-κB, and p38 MAPK . The ethrol diet . These sThe CW used in this study was grown in Jeju-island, Korea. Briefly, the dried powder of CW was extracted with 70% alcohol for 15 h at 60 °C to increase the cynandione A content and extraction yield. Thereafter, the CW powdered extract was concentrated under reduced pressure and lyophilized. The powdered extract was also prepared such that cynandione A, the main component of CW, was at a concentration of 15.0–22.5 mg/g. The test products were prepared to contain 25 or 50% CW ethanol extract powder. The treatment groups were divided into low-dose  and high-dose  groups. Participants were instructed to take two tablets once a day with water. The dose setting was based on a study in which total plasma cholesterol was significantly improved after the administration of 300 mg/kg body weight/day (BW/d) of CW for 4 weeks in an animal model of hyperlipidemia . The dos2, LDL-C≥ 130 mg/dL, and TG < 300 mg/dL. The following exclusion criteria were applied: LDL-C ≥ 190 mg/dL; intake of medicines and health functional foods that affected lipid and inflammatory metabolism within 4 weeks of the first visit; weight loss or weight change of 10% or more within 6 months of the first visit; patients suffering from myocardial infarction and stroke; patients with uncontrolled hypertension (systolic blood pressure ≥ 140 mmHg or diastolic blood pressure ≥ 100 mmHg) or diabetes (fasting blood glucose ≥ 180 mg/dL); patients with impaired hepatic function  or renal function (serum creatinine level ≥ 1.4 mg/dL); patients with chronic inflammatory bowel disease or autoimmune disease; patients with hyperthyroidism, hypothyroidism or malignancy; women under hormonal therapy; excessive smokers (≥20 cigarettes/day); alcoholic or drink more than 140 g/week; performing regular intense exercises (≥10 h/week); participation in another clinical trial within 4 weeks of the first visit; and hypersensitivity to CW.Adult men and women over 20 years of age were recruited at Seoul National University Hospital for this study. All recruited participants provided written informed consent and had BMIs between 18 and 30 kg/mThis was a double-blind, randomized, placebo-controlled, and parallel study. After a screening test, the eligible subjects were randomly assigned to the placebo, CWL, or CWH groups, and they consumed the placebo or test food for 8 weeks. Anthropometry and vital signs were measured at weeks 0, 4 and 8. Furthermore, the subjects were assessed for their history of drug and functional food intake. The subjects’ diets were maintained as usual from the run-in period until the end of this study, but some restrictions were in place: foods containing CW and medicines that affect the lipid metabolism in the body, as well as limiting their intake of foods with high cholesterol. The subjects recorded their daily intake of meals, snacks and drinks (excluding water) on a smartphone application developed by BiofoodCRO  for average dietary composition . The protocol was approved by the Institutional Review Board of Seoul National University (IRB No. H-1508-040-694). This study was also registered on the International Clinical Trials Registry Platform of the WHO .After the run-in and the intervention periods were completed, fasting blood was collected in Vacutainer blood collection tubes . The sera centrifuged in serum separation tubes were dispensed in 1.5 mL Eppendorf tubes and stored at −80 °C. Ethylenediaminetetraacetic acid (EDTA) tubes were used for plasma and peripheral blood mononuclear cell collection. First, the plasma was separated from the blood by centrifugation and then stored in 1.5 mL Eppendorf tubes at −80 °C. Peripheral blood mononuclear cells isolated from the buffy coat were washed with cold phosphate buffered saline and stored in RNA stabilization solution  at −80 °C. CETP and LCAT levels in the plasma were analyzed using an enzyme-linked immunosorbent assay (ELISA) kit .n = 28 per group), we referenced a human study that evaluated the efficacy of materials in improving cholesterol in blood [p < 0.05.For the purpose of calculating a sample size  completed the study . After 8The baseline characteristics of the 74 participants whose data were included in the statistical analyses are provided in r = −0.516, p = 0.010) and CWH  groups . The changes in LDL-C were significantly correlated with baseline LDL-C levels in the placebo , CWL , and CWH  groups  and CWH  groups (p = 0.027) (The correlation between baseline LDL-C levels and changes in lipid parameters after 8 weeks is shown in ) groups A, howeve) groups B. The ch) groups C. There ) groups D. The ch= 0.027) E.p = 0.022) and TG (p = 0.022) levels were higher in the CWL-treated group than in the placebo group and significantly reduced after 8 weeks. TC and LDL-C levels showed a decreasing trend after 8 weeks in both the high-level and low-level groups; however, there were no significant differences between the groups. Relative to the initial baseline LDL-C recorded, subjects were divided into two subgroups; high-level LDL-C ≥ 150 mg/dL (n = 33) and low-level LDL-C < 150 mg/dL (n = 41). In the high-level LDL-C subgroup, TC levels in the subjects treated with CWL (p = 0.012) or CWH (p = 0.021) were significantly reduced after 8 weeks compared to the levels in placebo-treated subjects (p = 0.036) were significantly different among the three groups. LDL-C levels in the CWL group (p = 0.032) were significantly reduced after 4 weeks, but there were no statistically significant differences until 8 weeks. However, in the low-level LDL-C subgroups, there were no other statistically significant differences between the groups (The lipid profiles of the participants are given in subjects B. In adde groups C.p = 0.022) and CWH-treated (p = 0.016) subjects were significantly reduced after 8 weeks compared to the levels in placebo controls. CETP was also significantly decreased in the CWL (p = 0.037) and CWH (p = 0.016) groups compared to that in the placebo group. In addition, CETP (p = 0.030) levels were significantly different among the three groups based on the group * time effect. In the low-level LDL-C subgroup, apoA1 levels were increased when treated with CWL (p = 0.028) compared to those in the placebo group. Furthermore, LCAT levels were significantly increased in the CWL group (p = 0.032).There were no significant differences in the levels of apo and cholesterol-related enzymes among the groups . HoweverIn a previous study, the ability of CW  to regulate blood cholesterol was evaluated in a hyperlipidemia rat model induced by a high-fat diet. The results showed that TG, VLDL-C and apoB were significantly reduced in CW groups. Furthermore, apoA1 was significantly increased in the CW group. These studies suggested the need for further evaluation of CW in randomized placebo-controlled trials and the possible development of CW supplements for the control of blood cholesterol in vivo.Similar to a previous animal study, a correlation analysis showed that subjects with higher baseline LDL-C levels benefited greater from CW treatment and significant changes in their lipid profiles was observed. Also, the results of this study showed that TG and VLDL-C levels are significantly reduced by the daily consumption of 300 mg CW for 8 weeks compared to levels in placebo-treated controls. Compared to a high dose, this study found a significant reduction in TG and VLDL-C only at a low dose. Many plant extracts showed hormesis which is the phenomenon where a specific chemical is able to induce biologically opposite effects at different doses ,29. In tNotable is the decrease in apoB, CETP and TC levels due to CW in subjects with baseline LDL-C levels ≥ 150 mg/dL. There was also a significant decrease in LDL-C levels. Similar to our results, consumption of green tea extract for 12 months significantly reduces TC and LDL-C levels in healthy postmenopausal women with baseline TC levels ≥ 200 mg/dL ,32. It h−/− mice [In subjects with lower LDL-C levels <150 mg/dL), LCAT and apoA showed a significant increase only in the CWL group when compared to the placebo group. The ethanolic extract of CW increases blood HDL-C levels and reduces the atherogenic index although there was no significant difference in total cholesterol in the high-cholesterol diet SD rats and apoE−/− mice . In this−/− mice ,18. Besi0 mg/dL, −/− mice . Among t−/− mice . PON1 bi−/− mice . A few d−/− mice ,43. This−/− mice . In addiThe correlation analysis also showed that changes in blood cholesterol due to CW intake were more effectively controlled in participants with LDL-C levels of 150 mg/dL or more than in those with slightly higher LDL levels than normal. TC levels decreased only in the subjects with higher LDL-C levels. Although LCAT and apoA1 increased significantly in the CWL group during subgroup analysis, HDL-C levels were not changed by CW supplementation. This suggests that CW might be effective in subjects with higher LDL levels. CVD, a major cause of death, has an important relationship with blood cholesterol, especially LDL-C levels. Previous studies have demonstrated a 20% reduction in CVD risk by lowering LDL-C levels to 38.67 mg/dL. Bjorck et al. concluded that the largest reduction in death was explained by substantial reductions in total cholesterol levels, resulting in a 39% of the mortality reduction ,46. CETPUnfortunately, although sample size was calculated based on a previous human study that evaluated the efficacy of materials in improving cholesterol in blood, statistical significance was not reached in the major endpoints such as TC or LDL-C levels by CW supplementation. A significant difference was found in the results only in stratification analysis, this might be due to the relatively small number of subjects in each subgroup as well as the relatively healthy blood cholesterol levels. Accordingly, clinical studies with higher blood cholesterol levels or LDL-C levels that are large enough to obtain statistical reliability are needed in the next study. In addition, because of the limitations associated with in vitro and animal studies, active compounds have not been identified. Nevertheless, this study might be worth noting because it is the first clinical study that evaluates the effect of CW on hyperlipidemia. This study also identified the safety of CWE supplementation as well as its efficacy. During the study, several adverse events were reported such as discomfort in urinary function, allergic reaction, vomiting, problems with skin, and gastrointestinal discomfort. All of these events were mild reactions and there was no statistical significance among treated groups. Our findings in this study show the possibility that CW may be developed as food supplements for helping maintain blood cholesterol levels in people with high levels of LDL cholesterol.


However, the complicated double-layer production of PAXIL® CR faces the risk of layer separation. In this study, PHH enteric coating single layer controlled-release tablets (PHH-EC-SLTs) were designed as a simplified substitution of PAXIL® CR through a rational formulation screening. The optimized PHH-EC-SLTs showed similar release behaviors in vitro to PAXIL® CR and the release profiles corresponded to a zero-order release model (R2 = 0.9958). Polymer matrix erosion was the main release mechanism, according to the fitting exponents n > 1 in the Korsmeyer-Pappas model. Crucial pharmacokinetic parameters including peak-reaching time (Tmax), peak concentration (Cmax) and the area under the blood level-time curve (AUC0-48) of PHH-EC-SLTs and PAXIL® CR had no significant difference (p > 0.05) and the relative bioavailability (F = 97.97%) of PHH-EC-SLTs demonstrated their similar pharmacokinetic profiles in vivo. In view of avoiding layer separation risk and simplifying the preparation processing, the self-made PHH-EC-SLTs could be considered as a safe and economic alternative to PAXIL® CR.Major depressive disorder (MDD) is one of the main contributors to disability and suicide mortality globally. Paroxetine hydrochloride (PHH) is the most potent antidepressant used for MDD treatment. Due to its reduced side effects PAXIL Major depressive disorders (MDDs) are common mental disorders diagnosed in nearly all age groups and all world regions . GloballMuch effort has been made to develop an effective therapy for MDD. Antidepressant administration was considered as an important treatment . Paroxet® CR is a controlled-release tablet produced by GlaxoSmithKline which adopted the patented Geomatrix® technology of Skypepharma. It can significantly reduce the abovementioned side effects [® CR finds wider application among the PHH formulations.Administration of conventional PHH tablets may induce nausea and lead to early treatment discontinuation due to the gastrointestinal reactions and the remarkable peak-valley fluctuation of its plasma concentration. PAXIL effects ,9, which effects . Thus, P® is an oral drug delivery system technology characterized by multilayer hydrogel matrix tablets, it is applicable to either poorly or highly soluble drugs and various drug release profiles can be obtained, including multiphase and zero-order release [® CR has a red block layer without drug and a white core layer with PHH as shown in ® CR from the white core layer, which shows zero-order release. The red block layer could decrease the contact between the drug and dissolution medium in the early stage to reduce the initial burst release. As time goes by, the red block layer is corroded gradually and the white core layer with PHH is swollen by water, so the surface area between PHH and dissolution medium is increased which would accelerate the PHH release in the later stage. Despite the advantages of PAXIL® CR, its preparation procedure is rather complicated, and therefore the risks of preparation failure and double-layer separation may occur. Considering the important function of the red block layer, the drug release behavior of PAXIL® CR would change and it would not achieve the desired zero-order release. The PHH release would also fail to be controlled if the two layers stick together insufficiently. These changes in release behavior may result in unknown and potentially dangerous side effects. Besides, the complicated preparation method also increases the production costs.Geomatrix release . Specifi® CR. That was because in the PHH-EC-SLTs, the drug release control was based on the properties of the composite gel skeleton (CGS) in the only layer of tablets without another helping layer. Previous studies showed that the CGS of hydroxypropylmethyl cellulose (HPMC) and sodium carboxymethyl cellulose (CMC-Na) or hydroxypropyl cellulose (HPC) and CMC-Na had a more stable sustained-release profile compared to the individual polymers [To solve the abovementioned problems, in this work PHH enteric coating single layer controlled-release tablets (PHH-EC-SLTs) were designed as an alternative of PAXILpolymers . We tookpolymers ,17. This2 factorial design [HPMC K4M and CMC-Na are water absorbing or swelling polymers which are commonly used in the preparation of sustained release tablets. In this work, based on the related literature and our previous study, HPMC K4M and CMC-Na were chosen as to formulate the CGS system. An accurate selection of the excipients is very important for the development of drug delivery systems . In ordel design ,20,21 wa® CR. Thus, the optimized PHH-EC-SLTs formulation developed in this work may be a prospective alternative to PAXIL® CR and will be beneficial for advancing the current therapy of MDD.The good product reproducibility of PHH-EC-SLTs suggests that the preparation technology is practical and repeatable. It was demonstrated that the self-made PHH-EC-SLTs with a simple preparation process had similar release behaviors in vitro and pharmacokinetic profiles in vivo compared to PAXILN-methyl-paroxetine were supplied by the National Institutes for Food and Drug Control . PAXIL® CR was purchased from GlaxoSmithKline plc.  and applied as the reference formulation. Clozapine was obtained from Shandong Qilu Pharmaceuticals Co., Ltd. . Methanol, acetonitrile and n-hexane were of chromatographic grade. All other chemicals used were of analytical grade. Beagle dogs  were purchased from Kangda Experimental Animal Technology Co., Ltd. . Dogs were maintained in separate cages and food and water were provided ad libitum.PHH was purchased from Zhejiang Linhai Jinqiao Chemical Co., Ltd. . HPMC K4M was obtained from Colorcon Co., Ltd. . CMC-Na was provided by Guangzhou Qihua Medical Equipment Co., Ltd. . Polyvinylpyrrolidone (PVP K30) was purchased from International Specialty Products Inc. . Microcrystalline cellulose (MCC) and magnesium stearate were purchased from Huzhou Pharmaceuticals Co., Ltd. . Lactose (Granulac 200) was obtained from Molkerei Meggle Wasserburg GmbH & Co. Kg. . Methacrylic resin (Eudragit L30D-55) was received from Evonik Röhm GmbH . The reference substance of PHH, desfluoroparoxetine and 2 factorial design, and other formulation and processing parameters of PHH-SLTs were determined by single factor experiments. Then, PHH-SLTs were coated to obtain PHH-EC-SLTs.PHH-SLTs were prepared with a CGS system composed of HPMC K4M and CMC-Na. The CGS system was optimized with a 32 factorial design was applied to optimize the content of the two critical excipients (HPMC K4M and CMC-Na). Their individual or combined effects on the formulation were evaluated. The compositions for the factorial design were summarized in A 3i was the number of drug release sampling, n was the total number of drug release sampling, tmid was the median time from i−1 to i, and ∆M was the increase of drug release from i−1 to i.Mean dissolution time (MDT) is the sum of different periods of time during which drug molecules or the dose stay in the polymer before release, divided by the total number of molecules or the total dose, respectively . It coulf2) was employed to investigate the similarity of different dissolution curves, and f2 was calculated according to Equation (2): Rt was the drug release rate of reference substance at time t, Tt was the release rate of specimen at time t, and n was the total number of drug release sampling. A high f2 value meant little differences between two formulations. In general, it was considered that two formulations have similar release behaviors in vitro if f2 ≥ 50.The dissolution curve of each formulation was compared with that of the reference formulation. Similarity factor  by SPSS 22.0 . They were considered significant when p < 0.01. Statistical results were obtained using Sigma Plot 13.0  to obtain three-dimensional response surface of f2 or MDT as a function of the amount of HPMC and CMC-Na:Statistical data was subjected to variance analysis (one-way ANOVA) and further binominal regression fitting with Equation (3) . Then, the sieved drug and excipients were precisely weighed and mixed homogeneously with appropriate volume of ethanol-water solution and then granulated by passing through 20# mesh (0.9 mm). Next, humid granules were dried in an oven at 40 °C, and then passed through 18# mesh (1.0 mm) to obtain uniform dry granules. Finally, the dry granules were mixed with magnesium stearate and silicon dioxide for the compressing procedure to prepare tablets.w/v). The whole preparation of PHH-EC-SLTs was illustrated by The formulation for enteric coating of PHH-SLTs was showed in Referring to the paddle method described in the general principles 0931 of Chinese Pharmacopoeia IV (2015) and considering the recommended dissolution method about PHH enteric coating controlled-release tablet by FDA, the method of in vitro release investigation was as follows: For the PHH-SLTs and PHH double layer controlled-release tablets (PHH-DLTs), they were cast into 1000 mL release medium of pH7.5 Tris-HCl (0.05 mol/L Tris added with 2 mol/L HCl to adjust the pH) and the rotating speed of paddle was 150 rpm. Furthermore, the temperature was controlled at 37.0 ± 0.5 °C. The concentration of PHH in release medium after 0.5, 1.0, 2.0, 4.0 and 6.0 h were measured and then release profiles were acquired. ® CR was different from above. The dissolution medium was 750 mL of pH 1.0 hydrochloric acid, and then replaced by pH 7.5 Tris-HCl. This was to simulate the circumstance of PHH in the stomach at the first 2 h and in the intestine at the following 6 h. The time intervals were 2.0, 2.5, 3.0, 4.0, 6.0 and 8.0 h.In vitro release investigation method of PHH-EC-SLTs and PAXILBesides, filter interference  and stab® CR were stored under the conditions of high temperature (60 °C), high humidity (RH 75 ± 5%) and strong light irradiation (4500 ± 500 lx), respectively. The appearance, content, related substance and release profiles in vitro after 0, 5 and 10 days storage were investigated.The PHH-EC-SLTs and reference preparation PAXIL® CR. Relevant data was summarized in ® CR for comparison studies of release mechanism with PHH-SLTs.Following related literatures, specifications and patents ,14,23,24Mathematic models would help to interpret the way drug release from the formulations. Four mathematic models including Zero-order, First-order, Higuchi and Korsmeyer-Peppas were fitted with the drug release profiles of PHH-SLTs and PHH-DLTs.w0) of PHH-SLTs and PHH-DLTs were weighed and added into the buffer solution (pH 7.5), the tablets were taken out 0.5, 1.0, 2.0 and 4.0 h later and water was removed carefully. Then, the wet mass and dry mass (drying at 50 °C) of the tablets were weighed. The water content and dry tablet mass were calculated by the Equations (4) and (5), respectively:Weight measurement was used to determine the dynamic water intake and weight loss by erosion. More specifically, the initial mass . Four beagle dogs were used for the in vivo pharmacokinetic study of PHH-EC-SLTs and PAXILv/v) and then assayed by HPLC.Blood samples were stored in an ice bath and centrifuged at 8000 rpm at 4 °C for at least 5 min. The harvested plasma was separated and stored at −20 °C pending analysis. Before the determination of PHH concentration, 0.5 mL methanol was added to 1 mL plasma to precipitate proteins, and the system was subjected to 8000 rpm centrifugation for 5 min. The supernatant was added with 0.1 mol/mL sodium hydroxide solution 0.2 mL to alkalify the solution. Moreover, 3 mL hexyl hydride was used to extract PHH. After 8000 rpm centrifugation for 5 min the upper organic phase was obtained and dried by nitrogen flow. The dried residue was dissolved in 100 µL of 80% methanol water solution ; mobile phase, 0.07 mol/L ammonium acetate-acetonitrile  which was adjusted the pH value to 5.5 by adding glacial acetic acid. Then, triethylamine was added in to prepare the final mobile phase 0.07 mol/L ammonium acetate-acetonitrile-triethylamine ; wavelength, 210 nm; column temperature, 40 °C; flow rate, 1.0 mL/min. Clozapine was used as the internal standard. Calibration was established within the range of 4.02~160.82 ng/mL. And calibration curves were obtained by performing a linear least-squares regression. No interference from endogenous plasma constituents was observed for the analyte or internal standards. The intra-day precision ranged from 0.10% to 3.19% with the accuracy ranging from 0.05% to 0.09%, respectively, and the deviation of inter-assay stability was 2.15%.Analysis of PHH concentrations in dog plasma was measured by HPLC analysis with an UltiMate 3000 HPLC system . The HPLC analysis conditions were: reversed phase C® CR were verified. Producing reproducibility and stability of PHH-EC-SLTs were also explored through comparing the release behaviors in vitro.The formulation of PHH-EC-SLTs was optimized by factorial design tests and single factor experiments. And the similarity of in vitro dissolution profiles between PHH-EC-SLTs and PAXILIt was presumed that the CGS system would achieve zero-order release in this work and HPMC K4M and CMC-Na were chosen to prepare the matrix core of PHH-SLTs. The hydration and strength of these two materials have a great influence on the drug release behavior of tablets made with them. On one hand, the hydration of HPMC K4M was much slower than that of CMC-Na. As a result, single HPMC K4M would have unsatisfactory release resistance capacity at the early stage, while the presence of CMC-Na would help to achieve desired controlled release behavior . On the 2 factorial design were prepared (f2 (Equation (2)) were obtained from the results of dissolution profiles, summarized in Factorial design would help to obtain the most appropriate content of HPMC K4M and CMC-Na. Nine formulations (F1~F9) of PHH-SLTs in the 3prepared  and the F) among nine formulations are shown in F value indicated that there were statistically significant differences (p < 0.01) among F1 to F9. Both contents of HPMC K4M and CMC-Na had significant influence on the drug release profiles of PHH-SLTs. Also, there was interaction between the content of HPMC K4M and CMC-Na.The variance analysis results  (p < 0.01), although the fitting degree value of f2 (F = 13.305) was less significant than the other four. As for the absolute value of b1 and b2 in Y60, Y120, and Y240, it was found that |b1| < |b2| in Y60 and Y120 while |b1| > |b2| in Y240. These results suggested that at the beginning of the drug release, the content of CMC-Na had greater influence than HPMC. However, this should be conversely applied to the case 2 h later. These results corresponded with the previous assumption that CMC-Na would compensate for the defects of HPMC K4M which could not maintain a steady drug release at the early stage (0~2 h) because of its much slower hydration rate, but the hydration of CMC-Na would be complete within 2 h, and then HPMC K4M would be the main water-absorbing polymer influencing the drug release and preventing the GCS system from breaking thanks to its high hydration gel strength over the whole release profile. This was why the CGS could avoid the disadvantages of single HPMC K4M and CMC-Na and achieve the desired controlled-release profile. Fully considering the fitting results of MDT and f2, it is reasonable to state that both HPMC and CMC-Na had a significant influence on the release behavior of the tablets. An appropriate content of HPMC K4M and CMC-Na could avoid their disadvantages and achieve desired controlled-release tablets.To understand the influence of HPMC K4M and CMC-Na on drug release, further binominal regression fitting (Equation (3)) was used. The calculating results were listed in f2 is displayed in f2. And it was revealed that 23% of HPMC and 24% of CMC-Na should be employed in the optimal formulation of PHH-SLTs. The 3D response surface for MDT and The content of HPMC and CMC-Na were ascertained based on the factorial design results. Different types of fillers and content of adhesives were also investigated to optimize the formulation of PHH-SLTs. Meanwhile, processing parameters including tableting method, tableting pressure and solvent were determined by single factor experiments. And the related results are shown in Lactose and MCC are frequently used fillers in the preparation of tablets. From The tablets prepared by a direct compression method would have a faster drug release in the early stage A. Unlikef2 = 62.49 > 50 when compared with the commercial formulation. This suggested that PHH-EC-SLTs had similar release behavior with PAXIL® CR (® CR in release behavior. The similar release behavior of three batches (f2 > 50) indicated the production of PHH-EC-SLTs were credible and reproducible  were used when comparing their release profile in vitro while the other parameters  were tested by comparing PHH-EC-SLTs and the commercial tablets. Because the PHH-EC-SLTs and PAXIL® CR utilized the patented Geomatrix® technology. Geomatrix ® is a technology for oral drug delivery system characterized by multilayer hydrogel matrix tablets [® is applicable to obtain multiphase and zero-order release profiles [® CR) had almost exactly the same drug release profile as PAXIL® CR ) could be used to describe drug release from polymeric systems which was the so-called power law : (6)MtM∞chanisms  and valuchanisms ,33,35. T® CR released PHH by polymer matrix erosion. However, the drug release during different periods might be quite different. The hydration and strength of HPMC K4M and CMC-Na had great influence on the drug release behavior of tablets made by the CGS system. Water absorption capacity and erosion weight loss of the CGS system when it was put into the dissolution medium could be its hydration and strength characteristics. Therefore, water absorption and erosion weight loss happening during the entire releasing processes were used to illustrate the hydration and strength changes of the CGS system, respectively. Thus, the release mechanism at a specific period of time would be better explained. It was demonstrated that both PHH-EC-SLTs and PAXILThe appearance of PHH-DLTs at different release points is shown in PHH-DLTs had a red block layer with no drug and a white core layer with PHH A. The blFrom 0.5 to 2 h, nearly the same water absorption rate of PHH-SLTs (about 27.45%) and PHH-DLTs (about 35.23%) were observed, although there was drastic difference in their erosion weight loss. These results suggested that the water absorption of CMC-Na reached saturation, and HPMC was the major water-absorbing polymer during this period. The fast erosion of the block layer contributed to the large amount of weight loss of PHH-DLTs. With the swelling of HPMC, the drug release mechanism was a combination of drug diffusion and matrix erosion. For PHH-SLTs, the powerful inter-molecular interaction between swelling HPMC and CMC-Na ensured the strong continuity of the matrix. When the hydrated gel layer was fully formed, the erosion rate of polymers became slower, but the drug diffusion grew faster because of high content of water, and the drug release mechanism was mostly affected by drug diffusion . During ® CR displayed similar in vivo pharmacokinetics profiles. The content of PHH in the plasma of beagles was analyzed at different time points. As shown in ® CR in vivo over time were identical on the whole.Ultimately, a pharmacokinetics investigation was performed in order to figure out if PHH-EC-SLTs and PAXIL® CR pharmacokinetic studies in humans [® CR and 35.67 ± 11.66 ng/mL for PHH-EC-SLTs). The PHH concentration increased to 50.33 ± 20.05 ng/mL for PAXIL® CR and 43.14 ± 15.69 ng/mL for PHH-EC-SLTs at 2h and then reduced to 27.80 ± 9.08 ng/mL for PAXIL® CR and 29.08 ± 4.99 ng/mL for PHH-EC-SLTs at 4 h. Although no pharmacokinetic studies of PHH enteric controlled-release tablets in beagles were reported, it was well documented that the destructive force in the dog stomach was evaluated to be 3.2 N, which was considerably higher than 1.9 N of humans [There was enteric coating for both group of tablets. Theoretically, no PHH release would be seen in the first 2 h (gastric acid condition), and this was confirmed by PAXILn humans ,30. Howef humans ,38. Hencf humans ,40. Howe® CR in beagles reached the maximum at 6 h, and a slow PHH concentration decline could be observed in the later period. The PHH concentrations of both PHH-EC-SLTs and PAXILTmax) and peak concentration (Cmax) were the tested results and are listed in the 0-48) and Cmax were engaged in variance analysis using SPSS22.0. The analysis results indicated that there was no significant difference between the vital parameters of PHH-EC-SLTs and PAXIL® CR (p > 0.05). The relative bioavailability of PHH-EC-SLTs towards PAXIL® CR was very high (F = 97.97%). To sum up, there were similar pharmacokinetics between PHH-EC-SLTs and PAXIL® CR in vivo.The pharmacokinetic parameters of each beagle were processed by WinNonlin 5.01  with a non-compartment model. Peak-reaching time (2 factorial design and single factor experiments were used to determine the optimal formulation of PHH-EC-SLTs. PHH-EC-SLTs possessed similar in vitro release behavior with PAXIL® CR. Meanwhile, the preparation process of PHH-EC-SLTs was reproducible, as inferred from the low batch variation. They underwent relatively little change when exposed to the unprotected outside environment, and they could be stored well in a cool and dry place if sealed. The release behavior of PHH-EC-SLTs and PAXIL® CR followed the zero-order equation and they mainly released the drug by polymer matrix erosion. Our release mechanism study showed that CMC-Na hydrated quickly to prevent rapid release of the encapsulated drug during the early stage, and the high gel strength of HPMC K4M could protect the CGS system from deformation. In addition, similar drug release in vivo and oral bioavailability between PHH-EC-SLTs and PAXIL® CR were also confirmed in our in vivo pharmacokinetics study. The results showed that jointly utilizing the different physicochemical property of polymers, hydration capacity and gel strength differences might be an advisable strategy for sustained-release tablet design and development.PHH-EC-SLTs were prepared based on the CGS system of HPMC and CMC-Na. The 3® CR. A 32 factorial design was applied to determine the optimal contents of HPMC and CMC-Na. And PHH-DLTs were used comparatively to study the release mechanism difference with PHH-SLTs. The results showed that there was similar drug release in vivo and oral bioavailability between PHH-EC-SLTs and PAXIL® CR in spite of different drug release mechanisms. In view of simplifying their processing and avoiding risk of double-layer separation, PHH-EC-SLTs could be considered as a prospective alternative of PAXIL® CR, and could be beneficial for advancing the current therapy of MDD.In this work, CGS materials were used to prepare the PHH-EC-SLTs, which imitated the drug release of PAXIL


Enterobacteriaceae are considered by WHO as “critical” priority pathogens for which novel antibiotics are urgently needed. The dissemination of carbapenemase-producing Escherichia coli (CP-Ec) in the community is a major public health concern. However, the global molecular epidemiology of CP-Ec isolates remains largely unknown as well as factors contributing to the acquisition of carbapenemase genes.Carbapenem-resistant E. coli sequence type (ST) 410 and its disseminated clade expressing the carbapenemase OXA-181. We reconstructed the phylogeny of 19 E. coli ST enriched in CP-Ec and corresponding to a total of 2026 non-redundant isolates. Using the EpiCs software, we determined the significance of the association between specific mutations and the acquisition of a carbapenemase gene and the most probable order of events. The impact of the identified mutations was assessed experimentally by genetic manipulations and phenotypic testing.We first analyzed the whole-genome sequence and the evolution of the ftsI encoding the penicillin-binding protein 3 and in the porin genes ompC and ompF. Mutated ftsI genes and a specific ompC allele related to that from ST38 inducing reduced susceptibility to diverse β-lactams spread across the species by recombination. We showed that these mutations precede in most cases the acquisition of a carbapenemase gene. The ompC allele from ST38 might have contributed to the selection of CP-Ec disseminated lineages within this ST. On the other hand, in the pandemic ST131 lineage, CP-Ec were not associated with mutations in ompC or ftsI and show no signs of dissemination.In 13 of the studied STs, acquisition of carbapenemase genes occurred in multidrug-resistant lineages characterized by a combination of mutations in Ec have started to disseminate globally. However, their selection is a multistep process involving mutations, recombination, acquisition of antibiotic resistance genes, and selection by β-lactams from diverse families. This process did not yet occur in the high-risk lineage ST131.Lineages of CP- Enterobacteriaceae (CRE) threatens the efficacy of current treatment options. Carbapenem resistance may result from a combination of mutations leading to reduced permeability  and overexpression of an extended-spectrum β-lactamase (ESBL) or a cephalosporinase that shows a weak activity against carbapenems [Enterobacteriaceae belong to Ambler class A (KPC-type), class B , or class D (OXA-48-like enzymes) [Escherichia coli, carbapenemase-producing E. coli (CP-Ec) might follow the same expansion and dissemination in hospitals and the community as the one observed for CTX-M-type ESBL-producing E. coli isolates [Antibiotic resistance is one of the most urgent public health concerns. The increasing rate in antimicrobial resistances worldwide suggests a bleak outlook in terms of morbidity, mortality, and economic loss . Carbapebapenems . Howeverbapenems . The majenzymes) . As thesisolates , 7, a scEc is complex with geographical diversity in terms of carbapenemase genes and of dominant lineages [Ec strains isolated in 16 countries between 2008 and 2013 revealed that 36% belonged to the pandemic ST131, which has driven the global spread of CTX-M-15 ESBL in E. coli [Ec was identified by the French National Reference Centre (Fr-NRC) between 2012 and 2013 [Ec clone carrying blaOXA-181 on an IncX3 plasmid. This lineage was predicted by a Bayesian analysis to have acquired blaOXA-181 around 2003 and subsequently blaNDM-5 around 2014 [The epidemiology of CP-lineages . Most stlineages , 8–14. H E. coli . SimilarEc lineages have not been explored. Here, by using an in-depth evolutionary and functional analysis of Ec ST410 and by extending it to the whole E. coli species, we show that acquisition of carbapenemase genes followed different evolutionary trajectories. In most STs, it occurred preferentially in specific disseminated lineages mutated in ftsI encoding penicillin-binding protein 3 (PBP3) and/or in the ompC and ompF porin genes. We also show that these mutations lead to a reduced susceptibility to some β-lactams including ertapenem. In phylogroup D and in particular in ST38, a specific ompC allele might explain the high prevalence of CP-Ec isolates within this lineage. On the other hand, we did not identify mutations in ftsI and ompC among ST131 isolates. These new data on the evolution of CP-Ec allow us to propose a model for their selection and dissemination.Despite public health implications, factors contributing to the emergence and the dissemination of CP-E. coli isolates analyzed in this work are listed in Additional file Ec ST410 isolates came from the strain collection of the Fr-NRC for antibiotic resistance. Four Ec ST410 clinical isolates came from the microbiological collection of the Public Health Faculty of the Lebanese University  and three ST410 isolates of animal origin from the ANSES strain collection. Test for OmpC permeability to β-lactams was performed in a W3110 derivative strain deleted for ompC and ompF genes [ompC ΔompF pOXA-232 strains carrying pACYC184 derivatives, disk diffusion assays were performed on Mueller Hinton (MH) agar plates supplemented with 2 mg/l chloramphenicol. Fitness was determined by growth curve analysis with an automatic spectrophotometer Tecan Infinite M200 during 24 h in LB, MH, or M9 media supplemented with 0.4% glucose. Growth metrics were estimated with the R package “growthcurver” [Features of the clinical pF genes . AntibiopF genes  or by Ethcurver” . The areEc genomes were sequenced by using the Illumina HiSeq2500 platform, with 100 nucleotide (nt) single-end reads for the four isolates from Lebanon and 100 nt paired-end reads for the other isolates. Libraries were constructed by using the Nextera XT kit (Illumina) following the manufacturer’s instructions. The OXA-181-producing Ec-MAD ST410 isolate was selected as a reference strain and sequenced to completion by using the long-read PacBio technology; 10,947 E. coli and 1451 Shigella genome sequences deposited in the NCBI database (19 June 2018) were retrieved for a global analysis of the specificity of CP-Ec . Ninety-six additional Ec ST167 isolates were retrieved from Enterobase (https://enterobase.warwick.ac.uk/). Raw reads from 62 Ec ST410 and 21 Ec ST38 isolates identified in Enterobase were retrieved from the NCBI database. . Graphs of genomic regions of interest were drawn with genoplotR [The PacBio reads were assembled with the RS_HGAP_Assembly.3 protocol from the SMRT analysis toolkit v2.3  and withenoplotR . For eacenoplotR , and theenoplotR , with anenoplotR , and theEc ST410 isolates, variants associated with different clades of the phylogeny were extracted with VCFtools [Sequence reads were mapped to reference genomes with BWA . For eacVCFtools  and annoVCFtools . The effVCFtools . The alghttp://tree.bio.ed.ac.uk/software/figtree/) and annotated trees with the script plotTree, R (https://github.com/katholt/plotTree).Pseudo-chromosomes were generated for each strain by parsing the pileup files created with SAMtools  using a ompC mutations” encompassing acquisition of ompC alleles from phylogroup D strains by recombination, ompC mutations changing the charge of AA in the pore lumen, and ompC inactivation; (ii) “ompF mutations” including ompF gene inactivation and point mutations in the OmpR binding sites of its promoter; (iii) “ftsI mutations” including the four different four-codon insertions  in ftsI; and (iv) the acquisition of a carbapenemase gene. We have focused our analysis on the independence between carbapenemase gene acquisition and mutation in each of the three genes, ompC, ompF, and ftsI.To assess the association between the different genetic events, we used the method developed by Behdenna et al.  implemenompC alleles and their regulatory regions were cloned into the medium copy number vector pACYC184 [ompC_Xba_F and ompC_Hind_R . The absence of mutation was checked by Sanger sequencing. Five different alleles were cloned into pACY184: wild-type (WT) MG1655 and the G137D mutated allele, the ST410 WT allele and the R195L mutated allele from Ec-MAD and the ST38 allele. Plasmids containing the ompC genes as well as the empty vector were introduced into competent W3110 ΔompC ΔompF pOXA-232. Competent cells were prepared by the CaCl2 method [Klebsiella pneumoniae isolate from the Bicêtre Hospital collection carrying this plasmid. Plasmid content in transformants was verified by plasmid DNA extraction (Qiagen) and Sanger sequencing.pACYC184  followin2 method . Plasmid2 method  was prepftsI gene of the MAD strain were reconstructed in a MG1655 genetic background to analyze their effects on antibiotic resistance. To this aim, we introduced the 12 nt insertion (YRIN form) and the 2 non-synonymous SNPs (E349K and I532L) into the E. coli strain MGF (MG1655strepR_F’tet_∆traD::Apra) by TM-MAGE [600 reached 0.6–0.7. The recombinase and Dam methylase were induced by the addition of l-arabinose  and further incubation for 10 min. Cultures were then chilled for 15 min on ice and centrifuged at 7300g at 4 °C. Two successive washes by 50 and 10 ml of cold water were performed, and the final pellet was resuspended in 200 μl water. One hundred microliters of cells was used for electroporation with 2 μl oligonucleotides Mut1ftsI or Mut2ftsI  or with the same volume of water as control and further incubated for 20 min. Bacterial pellets were collected and stored at − 80 °C. Total RNA was extracted with the Total RNA purification kit Norgen Biotek. cDNAs were synthetized from 500 ng of RNA with the Superscript II reverse transcriptase . Primer pairs were designed for the ompC and ompF genes, targeting divergent regions from these two genes, and for the reference gene recA . One-sided test was used for the comparison of the number of ARG, and two-sided test was used for the comparison on the area under the growth curve between the six isolates from the ST410 fluoroquinolone-resistant (FQR) clade.The statistical significance of the differences in the expression in qRT-PCR experiments was assessed by using a two-tailed Ec lineages, we first analyzed ST410 CP-Ec isolates, which show a high prevalence among isolates collected by the Fr-NRC [Ec isolates, 50 collected by the Fr-NRC (including 22 from patients repatriated from 15 different countries), 4 from Lebanon, and 3 non-CP isolates of animal origin  which corresponds to the previously described clade B4/H24RxC [Ec isolates not belonging to the OXA-181 subclade carry different carbapenemase genes from the OXA-48, KPC, VIM, and NDM families.In order to determine the genetic bases for the dissemination of CP-e Fr-NRC . We sequllection , with a 4/H24RxC . The 24 Ec-MAD). Ec-MAD carries 3 plasmids and 16 ARGs targeting 7 classes of antibiotics , the FQR-clade (n = 9), and the OXA-181 subclade (n = 16)  and one from the Fr-NRC (blaOXA-181); and isolated CP-Ec isolates  locus from ftsI to secM  were also observed in the other ST410 isolates recombined in the dcw region. These insertions resulting from a 4-codon duplication (YRIN) and from a subsequent mutation (YRIK) were first described in NDM-producing E. coli isolates from different STs [ftsI gene: E349K and I532L in association with the YRIN insertion and A413V with the YRIK insertion. The YRIK insertion in PBP3 was previously shown to confer reduced susceptibility to different β-lactams including ampicillin, cefepime, and aztreonam but not to carbapenems [One hundred ninety-seven SNPs including 16 non-synonymous (NS) mutations differentiated the common 16.5-kb region in the OXA-181 subclade from other rent STs . AdditioEc ST410 OXA-181 subclade, we analyzed the potential effect of non-synonymous mutations in the branch leading to its MRCA by using the SIFT algorithm [emrD and emrK and 5 from the class “cell envelope.” These mutations might have been selected in relation to modifications in antibiotic susceptibility.To identify additional polymorphisms that might have contributed to the dissemination of the lgorithm . We idenftsI mutation I532L; another affected the porin gene ompC at a conserved arginine residue in the L4 loop , one of the gateways for carbapenems  OmpR binding site. OmpR is a transcriptional activator of ompF and ompC expression, and this mutation is predicted to affect ompF expression  encoded a carbapenemase gene, and 248 (2.3%) carried a 4-AA insertion in PBP3: 163 YRIN, 49 YRIK, 3 YTIP, and 33 YRIP , ST167 , and ST405   or the YRIK (n = 2) insertion. The recombined regions differed by their lengths and by their patterns of polymorphisms. This indicated that they were due to independent events occurring at different positions in the phylogeny. Eleven of these recombination events affected the ancestor of at least 1 CP isolate. In particular, the same recombined region was shared by a subclade of 40 nr isolates carrying carbapenemase genes from 7 different types, suggesting that recombination occurred in the ancestor of the subclade before the acquisition of a carbapenemase gene. Only 5 out of the 54 Ec ST167 CP isolates did not undergo recombination at ftsI. Strikingly, we also observed likely recombination events internal to ST101 and ST156 where the initial YRIN or YRIP insertions were predicted to have occurred. Indeed, the same combinations YRIN/L532 or YRIP/V413 were detected in scattered lineages of ST101 and ST156, respectively, suggesting intra-ST recombination events , contains only the mutated ftsI gene with an YRIN insertion , no isolate was mutated in ftsI  out of 286 from the NCBI were mutated in ompF compared to only 138 (8%) out of the 1659 non-CP-Ec nr isolates. This reveals a likely association between ompF alterations and acquisition of a carbapenemase gene. In 89 CP-Ec nr isolates (31%), OmpC was modified, but in only 3 CP-Ec (1%) isolates, it was inactivated. In non-CP-Ec isolates, OmpC was modified in only 44 (3%) nr isolates and inactivated or missing in 39 (2%)  developed by Behdenna et al. . This mency Fig. b. In eacEc isolates), ST10 (12 nr-CP-Ec isolates), ST648 (11 nr-CP-Ec isolates), ST226 (7 nr-CP-Ec isolates), ST38 (26 nr-CP-Ec isolates), and ST69 (4 nr-CP-Ec isolates). ST648 and ST38 belong to phylogroup D, the phylogroup that served as the source for the dissemination of a specific ompC allele by recombination in other CP-Ec lineages. As this allele was present in the ancestors of the 2 STs and did not result from mutation or recombination during the evolution of the ST, it was not considered in our association analysis although it might confer a predisposition to acquire a carbapenemase gene. ST10 was the most numerous ST analyzed in this study with 528 nr isolates and showed a low rate of CP-Ec of 2% . Similarly, we observed a higher percentage of CTX-M enzymes among CP-Ec isolates compared to non-CP-Ec isolates except in ST131 and ST648 (from phylogroup D).A characteristic of the ST410 OXA-181 subclade compared to other ST410 ompC, ompF, and ftsI have been frequently selected in lineages that thereafter acquired carbapenemase genes by LGT. To further decipher the consequences of these mutations, we tested experimentally their impact on the susceptibility of E. coli to β-lactams. We first determined the contribution of the three mutations in ftsI  identified in the OXA-181 lineage. To this aim, we constructed derivatives of the ST10 strain MG1655 with combinations of these mutations  allele; and the WT ST410 allele and its R195L derivative were cloned into the medium copy number vector pACYC184 [ompC led to an increased susceptibility to β-lactams tested. However, we observed a differential effect of the different ompC alleles  between two isolates from the OXA-181 subclade (mutated) and two isolates from the FQR clade (non-mutated). As a control, we also quantified ompC expression. We observed a 15 to 30 and 5-fold reduction in ompF expression in LB and LB-NaCl respectively in the mutated isolates compared to wild type, whereas ompC expression remained unchanged , one isolate mutated in ftsI (YRIK insertion), and the two other isolates without any mutation in the three genes  of the non-redundant CP isolates analyzed in this work, a combined phylogenetic and statistical analysis revealed a significant association between mutations of these genes and subsequent acquisition of a carbapenemase gene  and mutations in ompK36 . In CP-Ec, we observed ompC inactivation in a few isolates, probably due to a high fitness cost of this event. Instead, the selected mutations affected ompC permeability towards β-lactams , and in 24 cases, it corresponded to disseminated lineages . We observed a similar situation for the porin OmpC, with 20 events of recombination of ompC alleles originating from phylogroup D isolates. The chromosomal region next to the ompC gene has been shown to be a hotspot of recombination [Neisseria spp. [E. coli, including carbapenems.During the evolution of CP-tes Fig. a. To ourisolates , 53. Thebination . Howeverbination  or in Neria spp. . AltogetblaOXA-181-bearing plasmid and blaCTX-M-15 gene with different patterns of mutations in ompC, ompF, and ftsI showed that in these clinical isolates and particularly in the broadly disseminated OXA-181 subclade, the increased resistance to β-lactams was not associated with a fitness cost , we did not observe any case of four AA insertions in ftsI or of mutations affecting AA in the pore lumen of OmpC among the 402 nr genome sequences we have analyzed. Furthermore, CP-Ec isolates were broadly distributed among the different ST131 lineages and were not associated with CTX-M type ESBL since only 37% also carried a β-lactamase gene of this class  according to the phylogenetic group among E. coli ST410 isolates. Figure S2. Recombination events in isolates of the E. coli ST410 FQR clade. Figure S3. Phylogenetic distribution of E. coli isolates mutated in ftsI. Figure S4. Recombination events at the dcw and ompC loci. Figure S5. Phylogeny and mutations in non-redundant E. coli ST131 CP-Ec isolates. Figure S6. Phylogeny and mutations in non-redundant CP-Ec isolates of ST48, ST206, ST224, ST359, ST361, ST448 and ST617. Figure S7. Phylogeny and mutations in non-redundant E. coli isolates of phylogroup D. Figure S8. Phylogeny and mutations of ST10, ST226, and ST746 non-redundant E. coli isolates. Figure S9. Estimated fitness of E. coli ST410 strains in rich and minimal media.Additional file 5: Table S9.ftsI, ompC and ompF mutations in STs encompassing CP-Ec isolates.Additional file 6:. Source data file of the work.